The goal of this application is to define the role of the Fas signaling pathway in the maintenance of peripheral tolerance in normal mice and in tolerance loss in the lpr/lpr murine model of systemic lupus erythematosus (SLE). The central tenet of these studies is that the principle site of Fas function in immune regulation is in the periphery, where a subpopulation of antigen-experienced T-cells express Fas and become susceptible to Fas-stimulated apoptotic suicide. Central to these studies will be the development of a new, peptide antigen-specific TCR transgenic lpr/lpr mouse model that will allow a more precise analysis of anergy and apoptotic deletion, and tolerance loss related to the Fas defect of the lpr phenotype. A double ab TCR transgene reactive with ovalbumin (OVA) in the context of I-Ad will be bred into lpr/lpr mice to provide a renewable source of Fas-deficient CD4+ T-cells of defined antigenic specificity. This system will allow careful study of Fas and CD28 regulation of tolerance mechanisms both in vivo and in vitro. In parallel, a panel of 'artificial' APCs is being developed that has controlled, constitutive expression of the class II MHC with or without coordinate expression of the counter-receptors for the CD28/CTLA-4 and Fas receptors. Sensitive techniques for in situ assay of apoptotic cell death will be coupled with a novel double-label in situ hybridization technique and flow cytometric analyses to perform single-cell analyses of T-cell populations manipulated in vitro and in vivo.